The stick of Cam Atkinson slid through the goalie mask as Holtby raised it to prepare to deflect a potential shot, and the stick appeared to make contact with Holtby near his eyes.
Holtby was treated on the bench before heading to the locker room. The Capitals announced during the second intermission that he would not return to the game.
Coach Todd Reirden was "very concerned'' and remained uncertain about Holtby's status afterward.
"He'll be getting evaluated later tonight still,'' Reirden said. "Our initial discussions with our trainer and doctors, that's a serious thing. He wasn't able to return to the game, and we'll get some more evaluation tonight and know a little bit more tomorrow and have a better update for you guys then.''
Atkinson, who scored earlier in the game, received a two-minute penalty for high sticking.
The Capitals had already gotten a scare when Alex Ovechkin left the game in the first period, but the NHL's leading goal-scorer came back for the second. Reirden said Ovechkin needed medical attention to close a cut on one of his hands.
The Associated Press contributed to this report.